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Phage Display Technology for Human Monoclonal Antibodies

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Human Monoclonal Antibodies

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1060))

Abstract

During the last 15 years in vitro technologies opened powerful routes to combine the generation of large libraries together with fast selection procedures to identify lead candidates. One of the commonest methods is based on the use filamentous phages. Antibodies (Abs) can be displayed successfully on the surface of phage by fusing the coding sequence of the antibody variable (V) regions to the phage minor coat protein pIII. By creating large libraries, antibodies with affinities comparable to those obtained using traditional hybridomas technology can be selected by a series of cycles of selection on antigen. As in this system antibody genes are cloned simultaneously with selection they can be easily further engineered for example by increasing their affinity (to levels unobtainable in the immune system), modulating their specificity or their effector function (by recloning into a full-length immunoglobulin scaffold). This chapter describes the basic protocols for antibody library construction, handling, and selection.

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References

  1. Marks JD et al (1991) By-passing immunization. Human antibodies from V-gene libraries displayed on phage. J Mol Biol 222(3):581–597

    Article  CAS  Google Scholar 

  2. Scott JK, Smith GP (1990) Searching for peptide ligands with an epitope library. Science 249(4967):386–390

    Article  CAS  Google Scholar 

  3. Boder ET, Midelfort KS, Wittrup KD (2000) Directed evolution of antibody fragments with monovalent femtomolar antigen-binding affinity. Proc Natl Acad Sci USA 97(20):10701–10705

    Article  CAS  Google Scholar 

  4. Boder ET, Wittrup KD (1997) Yeast surface dispay for screening combinatorial polypeptide libraries. Nat Biotechnol 15:553–557

    Article  CAS  Google Scholar 

  5. Smith GP (1985) Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface. Science 228(4705):1315–1317

    Article  CAS  Google Scholar 

  6. Bradbury A et al (2003) Antibodies in proteomics I: generating antibodies. Trends Biotechnol 21(6):275–281

    Article  CAS  Google Scholar 

  7. McCafferty J et al (1990) Phage antibodies: filamentous phage displaying antibody variable domains. Nature 348(6301):552–554

    Article  CAS  Google Scholar 

  8. Hoogenboom HR et al (1991) Multi-subunit proteins on the surface of filamentous phage: methodologies for displaying antibody (Fab) heavy and light chains. Nucleic Acids Res 19(15):4133–4137

    Article  CAS  Google Scholar 

  9. Sblattero D, Bradbury A (1998) A definitive set of oligonucleotide primers for amplifying human V regions. Immunotechnology 3(4):271–278

    Article  CAS  Google Scholar 

  10. Vaughan TJ et al (1996) Human antibodies with sub-nanomolar affinities isolated from a large non-immunized phage display library [see comments]. Nat Biotechnol 14(3):309–314

    Article  CAS  Google Scholar 

  11. Sblattero D, Bradbury A (2000) Exploiting recombination in single bacteria to make large phage antibody libraries. Nat Biotechnol 18(1):75–80

    Article  CAS  Google Scholar 

  12. Hoogenboom HR, Winter G (1992) By-passing immunisation. Human antibodies from synthetic repertoires of germline VH gene segments rearranged in vitro. J Mol Biol 227(2):381–388

    Article  CAS  Google Scholar 

  13. Sidhu SS, Kossiakoff AA (2007) Exploring and designing protein function with restricted diversity. Curr Opin Chem Biol 11(3):347–354

    Article  CAS  Google Scholar 

  14. Fellouse FA et al (2007) High-throughput generation of synthetic antibodies from highly functional minimalist phage-displayed libraries. J Mol Biol 373(4):924–940

    Article  CAS  Google Scholar 

  15. Krebs B et al (2001) High-throughput generation and engineering of recombinant human antibodies. J Immunol Methods 254(1–2):67–84

    Article  CAS  Google Scholar 

  16. Bradbury A et al (2003) Antibodies in proteomics II: screening, high-throughput characterization and downstream applications. Trends Biotechnol 21(7):312–317

    Article  CAS  Google Scholar 

  17. Di Niro R et al (2010) Rapid interactome profiling by massive sequencing. Nucleic Acids Res 38(9):e110

    Article  Google Scholar 

  18. Lim TS et al (2010) V-gene amplification revisited—an optimised procedure for amplification of rearranged human antibody genes of different isotypes. N Biotechnol 27(2):108–117

    Article  CAS  Google Scholar 

  19. Krebber A et al (1997) Reliable cloning of functional antibody variable domains from hybridomas and spleen cell repertoires employing a reengineered phage display system. J Immunol Methods 201:35–55

    Article  CAS  Google Scholar 

  20. Yamamoto KR et al (1970) Rapid bacteriophage sedimentation in the presence of polyethylene glycol and its application to large-scale virus purification. Virology 40(3):734–744

    Article  CAS  Google Scholar 

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Acknowledgments

This work was supported in part by Fondazione Cariplo, Compagnia Sanpaolo, NIH U54 DK093500-01. EC Marie Curie Research Training Network [contract no. MRTN-CT-20010-289964].

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Deantonio, C., Cotella, D., Macor, P., Santoro, C., Sblattero, D. (2014). Phage Display Technology for Human Monoclonal Antibodies. In: Steinitz, M. (eds) Human Monoclonal Antibodies. Methods in Molecular Biology, vol 1060. Humana, Totowa, NJ. https://doi.org/10.1007/978-1-62703-586-6_14

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  • DOI: https://doi.org/10.1007/978-1-62703-586-6_14

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  • Publisher Name: Humana, Totowa, NJ

  • Print ISBN: 978-1-62703-585-9

  • Online ISBN: 978-1-62703-586-6

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